Materials and methods for evaluating and treating neuromyelitis optica (nmo)

ABSTRACT

The invention provides prognostic methods for evaluating the severity of NMO and NMO-associated diseases as well as methods of treating NMO and NMO-associated diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/862,139, filed Jan. 4, 2018, which is a continuation of U.S. patentapplication Ser. No. 12/573,942 (now U.S. Pat. No. 9,891,219), filedOct. 6, 2009, which claims priority under 35 U.S.C. § 119(e) of U.S.Application No. 61/104,621, filed Oct. 10, 2008.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NS049577 awardedby National Institutes of Health. The government has certain rights inthe invention.

TECHNICAL FIELD

This disclosure generally relates to autoimmune diseases.

BACKGROUND

Neuromyelitis optica (NMO) is currently the best defined acquiredinflammatory demyelinating disorder of the central nervous system (CNS).NMO attacks optic nerves and spinal cord selectively and repeatedly.Clinical, histopathological and immunobiological observations support apathogenic role for an IgG autoantibody specific for the astrocyticwater channel aquaporin-4 (AQP4), and the severity of acute NMO isameliorated by antibody-depleting therapies.

In contrast to most inflammatory CNS demyelinating disorders, tissuedestruction in NMO is profound. In addition to white matter lesions, NMOcharacteristically exhibits central necrosis of spinal cord gray matter.Histopathological CNS lesions lack AQP4 and show deposition of IgM andIgG and products of complement activation in a vasculocentric patternthat coincides with the normal distribution of AQP4.

Until recently, NMO was considered a rare and severe variant of multiplesclerosis (MS). However, the advent of serological testing for AQP4-IgGhas revealed that NMO and its inaugural forms are more common thanpreviously recognized. They tend to be misdiagnosed as MS, which lacks aspecific biomarker.

SUMMARY

This disclosure provides prognostic methods for evaluating the severityof NMO and NMO-associated diseases as well as methods of treating NMOand NMO-associated diseases.

In one aspect, methods of providing a prognosis for an individual thathas NMO or a NMO-associated disease are provided. Such methods typicallyinclude providing a biological sample from the individual; anddetermining, in vitro, whether or not the biological sample reduces cellsurface expression of EAAT2 or reduces uptake of extracellular glutamatecompared to a biological sample from an individual that does not haveNMO or a NMO-associated disease. Typically, a reduction in cell surfaceexpression of EAAT2 or a reduction in uptake of extracellular glutamatecorrelates with a prognosis of the individual.

In another aspect, methods of providing a prognosis for an individualthat has NMO or a NMO-associated disease are provided. Such methodsgenerally include providing a biological sample from the individual;contacting the biological sample with primary astrocytes, adifferentiated astrocyte-type cell, or a non-astrocytic cell expressinga gene encoding aquaporin-4 or a functional fragment thereof; anddetermining, in vitro, whether or not the biological sample reduces cellsurface expression of EAAT2 or reduces uptake of extracellular glutamatecompared to a biological sample from an individual that does not haveNMO or a NMO-associated disease. Generally, a reduction in cell surfaceexpression of EAAT2 or a reduction in uptake of extracellular glutamatecorrelates with a prognosis of the individual.

Representative biological samples include serum, plasma, cerebrospinalfluid (CSF), and immunoglobulins.

In still another aspect, methods of providing a prognosis for anindividual that has NMO or a NMO-associated disease are provided. Suchmethods typically include providing a biological sample from theindividual; contacting the biological sample with cells or tissues inthe presence of aquaporin-4 polypeptides or functional fragmentsthereof; and determining whether or not complement is activated in thecells. Generally, an activation of complement correlates with aprognosis of the individual. In one embodiment, the aquaporin-4polypeptides or functional fragments thereof are expressed by the cells.

In one aspect, methods of treating an individual that has NMO areprovided. Such methods can include administering a glutamate receptorantagonist to the individual. Representative glutamate receptorantagonists include 1-amino-3,5-dimethyl-adamantane, 1-aminoadamantane,(+)-3-methoxy-17-methyl-(9α,13α,14α)-morphinan,17-methyl-9a,13a,14a-morphinan-3-ol,2-(2-chlorophenyl)-2-methylamino-cyclohexan-1-one,1-(1-phenylcyclohexyl) piperidine,(±)cis-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl) cyclohexanolhydrochloride, and 6-(Dimethylamino)-4,4-diphenylheptan-3-one. Suchmethods can further include administering a compound to the individualthat inhibits complement.

In still another aspect, methods of treating an individual that has NMOare provided. Such methods typically include administering a compound tothe individual that inhibits complement. Representative compounds thatinhibit complement include Compstatin, APT070 (Mirococept), solublecomplement receptor 1 (sCR1), anti-C5 antibody, eculizumab (Soliris®),and substituted dihydrobenzofurans, spirobenzofuran-2(3H)-cycloalkanes,and their open chain intermediates. Such methods can further includeadministering a glutamate receptor antagonist to the individual.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the quantitation of membrane cytotoxicityinduced by NMO serum and active complement (30 minutes at 37° C.).Results were the average of seven independent experiments each using anindividual serum pool from 10-15 different NMO patients or fromapproximately 350 control patients with miscellaneous disorders.

FIGS. 2A and 2B are graphs that demonstrate that, in primary astrocytes,NMO-IgG impaired glutamate uptake or complement activation. (A)Quantitation of membrane permeability after exposure to control or NMOserum. Increase in permeability to propidium iodide (PI)>2 fold by NMOserum required active complement (A C′=inactivated complement). (B)Uptake of L-[³H]glutamate (±Na+-containing buffer) without human serum(open box) or in control (shaded box) or NMO serum (hatched box). Excessunlabeled glutamate (dark box) prevented L-[³H]glutamate uptake. NMOserum reduced L-[³H]glutamate uptake by 50%. Experiment (B) wasperformed twice. All others were performed at least 3 times.

FIG. 3 demonstrates that expression of aquaporin proteins in HEK-293cells induced EAAT2 protein expression. Glutamate transport: GFP-AQP4cells took up approximately 3 fold more L-[³H]glutamate thanvector-transfected cells (note Na+-dependence). All experiments wereperformed a minimum of two times.

FIG. 4 shows a flow-chart showing pathways for serological evaluation ofpatients.

DETAILED DESCRIPTION

NMO-IgG is a clinically validated serum biomarker that distinguishesrelapsing CNS inflammatory demyelinating disorders related toneuromyelitis optica (NMO) from multiple sclerosis (MS). Thisautoantibody targets astrocytic aquaporin-4 (AQP4) water channels.Characteristic CNS lesions exhibit selective depletion of AQP4, with andwithout associated myelin loss, focal vasculocentric deposits of IgG IgMand complement, prominent edema and inflammation.

A marked reduction of the astrocytic Na+-dependent excitatory amino acidtransporter, EAAT2 (a homolog of rodent GLT-1 (Zeng et al., 2007, Mol.Cell. Neurosci., 34:34-9) in AQP4-deficient regions of NMO patientspinal cord lesions is described. Thus, binding of NMO-IgG to astrocyticAQP4 appears to initiate several potentially neuropathogenic mechanisms:complement activation and AQP4 and EAAT2 down-regulation. Since EAAT2accounts for >90% of glutamate uptake in the CNS, is critical forclearing glutamate from excitatory synapses and is expressed selectivelyin astrocytes, altered expression of EAAT2 can impair glutamatehomeostasis, resulting in over-stimulation of glutamate receptors inneurons and oligodendrocytes. This disruption in glutamate homeostasismay contribute indirectly to the pathobiology of NMO or NMO-associateddiseases. See, for example, Hinson et al. 2007, Neurology, 69:2221-31.

NMO and NMO-associated diseases encompass a number of neurologicaldisorders related to AQP4 autoimmunity. For example, NMO diseases arerepresented by a spectrum of adult or pediatric inflammatory CNSdemyelinating disorders related to AQP4 autoimmunity involvinglongitudinally extensive transverse myelitis, optic neuritis (e.g.,relapsing optic neuritis) or brainstem encephalitis. RepresentativeNMO-associated diseases include connective tissue disorders related toAQP4 autoimmunity (e.g., Lupus or Sjogrens syndrome) and cancers relatedto AQP4 autoimmunity (e.g., breast).

Prognostics

The relationship described herein between aquaporin-4 and EAAT2 and theassociated loss of EAAT2 in NMO or NMO-associated diseases can be usedin the prognosis of an individual. For example, the amount of reductionin cell surface expression of EAAT2 or the amount of reduction in theuptake of extracellular glutamate can be correlated with the severity ofthe NMO or NMO-associated disease or the accompanying symptoms.

Well known in vitro bioassays can be used to evaluate whether or not areduction in cell surface expression of EAAT2 or uptake of extracellularglutamate is observed. For example, a biological sample from anindividual can be contacted with primary astrocytes, differentiatedastrocyte-type cells, or non-astrocytic cells expressing a gene encodingaquaporin-4 or a functional fragment thereof, and either or both cellsurface expression of EAAT2 or the uptake of extracellular glutamate bythe cells can be evaluated. Cell surface expression can be evaluatedusing well known immunoassays such as innumohistochemistry or Westernblot (Immunoassay, 1996, Diamandis & Christopoulos, Eds., AcademicPress), and glutamate uptake by a cell can be evaluated, for example,using methods such as those described by Lin et al., 2001, Nature,410:84-8.

A reduction in the cell surface expression of EAAT2 or a reduction inthe uptake of extracellular glutamate (compared to the cell surfaceexpression of EAAT2 or the glutamate uptake that occurs in the presenceof a biological sample from an individual that does not have NMO or aNMO-associated disease) can be used prognostically. Typically, theamount of reduction in either or both the cell surface expression ofEAAT2 and the uptake of extracellular glutamate is directly related tothe severity of the NMO or NMO-associated disease that the individualwill experience.

In addition, the involvement of complement activation in NMO andNMO-associated diseases can be used in the prognosis of an individualsuffering from NMO or a NMO-associated disease. For example, the extentor degree that complement is activated in the presence of serum (orcomponents therein) from an individual from an individual that has NMOor a NMO-associated disease (compared to the extent or degree thatcomplement is activated in the presence of serum (or components therein)from an individual that does not suffer from NMO or a NMO-associateddisease) can be correlated with the severity of disease.

Methods for evaluating whether or not complement is activated in thepresence of a biological sample from an individual are known in the art.Such methods include, without limitation, exposing the biological sampleto cells or tissues in the presence of aquaporin-4 polypeptides (orfunctional fragments thereof), and determining whether or not complementis activated in the cells. For example, activation of complement bycells can be determined by evaluating cells for propidium iodide uptake(Kasibhatla et al., 2006, Cold Spring Harb. Protoc.; doi:10.1101/pdb.prot4495) or using immunoassays (e.g., immunohistochemistry)to detect the deposition or accumulation of one or more components ofcomplement (e.g., C9neo assembly or C5 deposition).

Any number of biological samples can be used in the methods describedherein. For example, a biological sample can include, withoutlimitation, serum, plasma, and cerebrospinal fluid (CSF). In addition, abiological sample can include immunoglobulins (e.g., engineeredimmunoglobulins or immunoglobulins that are secreted by cultured Blymphocytes or plasma cells). Alternatively, under autopsy conditions,biological samples can include tissue biopsies such as from spinal cordand brain.

It would be understood by those of skill in the art that, underconditions described herein in which aquaporin-4 polypeptides orfunctional fragments are required, such polypeptides or fragments can benaturally present in the cells (e.g., endogenously expressed or native),or cells can be genetically engineered to express aquaporin-4polypeptides or functional fragments thereof. In addition, aquaporin-4polypeptides or functional fragments thereof (e.g., purified aquaporin-4or functional fragments thereof) can be added exogenously to the cells,to the biological sample, or to a combination of the two.

Thereapeutics

The relationship described herein between EAAT2 and/or complementactivation with NMO-IgG provides a number of novel therapies fortreating NMO or NMO-associated diseases. For example, in some instances,an individual can be administered an effective amount of an antagonistof glutamate receptors can be administered to an individual. In someinstances, the antagonists are specific for glutamate receptors, and inother instances, the antagonists are specific for glutamate receptors onneurons, oligodendrocytes and astrocytes. An effective amount of anantagonist of glutamate receptors is an amount that reduces oreliminates the neurological effects caused by the accumulation ofextracellular glutamate.

Antagonists or glutamate receptors, also referred to as glutamatereceptor blockers, include, without limitation,1-amino-3,5-dimethyl-adamantane (Memantine®), 1-aminoadamantane(Amantadine®), (+)-3-methoxy-17-methyl-(9α,13α,14α)-morphinan(Dextromethorphan®), 17-methyl-9a,13a,14a-morphinan-3-ol (Dextrorphan®),2-(2-chlorophenyl)-2-methylamino-cyclohexan-1-one (Ketamine®),1-(1-phenylcyclohexyl) piperidine (Phencyclidine®),(±)cis-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl) cyclohexanolhydrochloride (Tramadol®), and6-(Dimethylamino)-4,4-diphenylheptan-3-one (Methadone®). Therapeuticagents also include, for example, agents that reduce the extracellularglutamate concentration such as, without limitation, soluble EAAT2receptor or antibodies that specifically recognize and bind to glutamate(e.g., anti-glutamate Ab).

In certain instances, an effective amount of a compound that inhibitscomplement or the activation of complement can be administered to anindividual. Compounds that inhibit complement are known in the art andinclude, without limitation, Compstatin, APT070 (Mirococept), solublecomplement receptor 1 (sCR1), anti-C5 antibody, eculizumab (Soliris®),and substituted dihydrobenzofurans, spirobenzofuran-2(3H)-cycloalkanes,and their open chain intermediates (see, for example, U.S. Pat. No.5,506,247).

In some instances, effective amounts of one or more antagonists ofglutamate receptors and one or more compounds that inhibit complementcan be administered to an individual. One or more antagonists ofglutamate receptors and one or more compounds that inhibit complementcan be administered simultaneously, or the one or more antagonists ofglutamate receptors and the one or more inhibitors of complement can beadministered sequentially. In certain instances, an antagonist ofglutamate receptors and a compound that inhibits complement can beadministered alternatively to an individual.

Routes of administering compositions to an individual are well known inthe art and include, for example, parenteral, e.g., intravenous,intradermal, subcutaneous, oral (e.g., ingestion or inhalation),transdermal (topical), transmucosal, and rectal administration. Acomposition for administering to an individual typically is formulatedto be compatible with its intended route of administration.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Part A Example 1—Cell Lines and Transgenic Constructs

Major binding sites of NMO-IgG (and affinity-purified AQP4-specificrabbit IgG) in sections of normal adult human and mouse CNS tissues arewhere AQP4 is expressed in highest density, namely in the dystroglycancomplex of the highly polarized plasma membrane of astrocytic footprocesses. The reason AQP4 is readily visualized at those sitesimmunohistochemically is because of its ultrastructural organization atthose sites in tightly packed arrays. In preliminary Western blotstudies (with protein overloaded conditions), it was observed that therat oligodendroglial-astrocytic progenitor cell line (CG4), whendifferentiated in vitro to astrocytic phenotype (stellate and expressingprominent cytoplasmic GFAP intermediate filaments), express minimal AQP4immunoreactivity, and that two independent human astrocytoma cell lines(HTB-14 and CRL-17; obtained from ATCC) did not express detectable AQP4by immunofluorescence or Western blot. Therefore, as the initial systemto investigate in vitro the immunobiological consequences of IgGinteracting with extracellular epitopes of AQP4, transfected humanembryonic kidney cell line (HEK293) over-expressing full-length humanAQP4 protein (fused at its N-terminus with green fluorescent protein[GFP]) was used. The parental HEK293 cell line does not express AQP4constitutively, but it does express dystroglycan complex partnerproteins of AQP4, which assure stable AQP4 insertion in the plasmamembrane. Furthermore, the proximity of adjacent AQP4 homotetramers inthe transfected cell's membrane mimics the close packing of epitopes inthe highly polarized astrocytic foot process.

Example 2—Antibodies and Sera

Fluorochrome-conjugated goat IgGs were purchased from Molecular Probes(Alex-Fluor 546; human, mouse or rabbit IgG-specific, and Oregon Green;mouse IgG-specific) or Invitrogen (Cy5; rabbit IgG-specific). Conjugatedmonoclonal mouse IgGs (FITC; specific for human IgG subclasses, or Cy3;specific for glial fibrillary acidic protein and sodium channel) wereobtained from Sigma, and unconjugated were obtained from BD Biosciences(Casprl and early endosome antigen-1 [EEA1]) and Dako, Denmark (humanCD138). Goat IgGs monospecific for human IgG (TRITC-conjugated) or humanIgM (FITC-conjugated) were from Southern Biotechnology. Rabbit IgGspecific for AQP4 residues 249-323 was obtained from Sigma. Rabbit IgGspecific for human C9neo was a gift from Dr. Paul Morgan (Cardiff, UK).Deidentified patients' sera were obtained from the NeuroimmunologyLaboratory, Department of Laboratory Medicine and Pathology, Mayo ClinicRochester, Minn.

Example 3—Immunostaining

All final preparations of live cells and sections of mouse tissue andautopsied human lymph node and brain tissues were mounted in ProLong®Gold DAPI antifade medium (Molecular Probes). Fluorescent images werecaptured from a Zeiss LSM510 confocal microscope.Immunoperoxidase-stained sections were counterstained with hematoxylinand photographed from an Olympus DP-BSW microscope and DP70 digitalcamera system.

Live cells were exposed sequentially to 20% human serum or rabbitanti-AQP4 IgG diluted 1:1000, secondary antibody (1:500) and chilled 95%ethanol/5% acetic acid (15 minutes). Cells fixed in 10% formalin werepermeabilized, blocked, and incubated in primary and secondary antibodyprobes. Tissues dissected from perfused mice were post-fixed overnightin 4% paraformaldehyde, held 24 hours in 30% sucrose, frozen in OCTmedium, sectioned (8 μm) and air-dried, incubated sequentially in 10%normal goat serum containing 0.1% Triton X-100 (30 minutes), primaryantibody (18 hours) and secondary antibody (1 hour; room temperature).Autopsied human lymph node and brain tissues, fixed in 10% formalin andembedded in paraffin, were deparaffinized, sectioned (5 μm), washed inbi-distilled water and steam-heated. Sections were incubated with 10%normal goat serum (1 hour) and then overnight at 4° C. with primaryantibody (anti-CD138 at 1:50; anti-IgG at 1:750; anti-IgM at 1:200).

Example 4—Antigen Modulation Assay

Complement was inactivated in the serum of patients and control subjectsby incubating the serum for 30 minutes at 56° C. Time-lapsephotomicrographs of living cells were captured at 15 minute intervalsfor 24 hours. Media containing human serum were replaced at 16 hourswith fresh media, with or without cycloheximide (0.25 μg/mL). Test serawere added to the culture media for either 16 hours at 37° C. or 1 hourat 4° C. Cells were washed, exposed 30 minutes to Alexa-Fluor546-labeled anti-human IgG (1:500), washed again, fixed in chilled 95%ethanol/5% acetic acid, rinsed in PBS, and stained with Hoescht (1μg/mL).

Example 5—Complement Activation Assays

GFP-AQP4-HEK 293 cells were incubated for 2 hours on ice with 20%heat-inactivated control or NMO patients' serum. Cells were washed withchilled DMEM media and exposed to 20% fresh (or heat-inactivated) normalhuman serum (as complement source) for 45 minutes at 37° C. Afterrinsing and fixing (10% formalin, 4 minutes), cells were incubatedsequentially at room temperature in 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (1 minute), 10% normal goat serumand rabbit anti-human C9neo antibody (30 minutes), washed and incubatedin labelled anti-rabbit IgG. To evaluate complement's effects onmembrane integrity, test pools of human serum were added to the cells at4° C. (for 1 hour). After subsequent incubation for 90 minutes at 37° C.(95% air/5% CO₂) in fresh or heat-inactivated Low-Tox-H rabbitcomplement (Cedarlane Lab; 20% final concentration), the monolayer wasexamined and photographed by transmitted bright field imaging. Toquantitate complement-mediated membranolytic activity by flow cytometricanalysis after sequential exposure to serum (30 minutes at roomtemperature) and complement (37° C. for 30 minutes), the cells werelifted with trypsin, washed and suspended in chilled buffer (10 mMHepes/NaOH pH 7.4; 140 mM NaCl; 2.5 mM CaCl₂) containing 0.5 μgpropidium iodide (BD Biosciences) and held in the dark for 15 minutesbefore analysis. Fold increase in membrane permeability was determinedby comparing the percentage of propidium iodide permeable GFP-positivecells to propidium iodide-permeable GFP-negative cells. In the absenceof antigen-specific activation of complement, the fold increase inmembrane permeability was less than 1.25 (125%).

Example 6—IgG Depletion and Purification

IgG was depleted from serum pools using protein G-agarose beads (2 hoursat 4° C.). IgG-depleted serum was used at a concentration of 20% in thecomplement assay. IgG eluted from washed protein G beads in 0.1 M aceticacid was neutralized, dialyzed against PBS and concentrated usingCentricon YM-3 filter devices (Millipore). IgG concentrations weredetermined by rate nephelometry.

Example 7—Time-Lapse Photomicrographs

GFP-AQP4-HEK293 cells were seeded onto optical quality glass bottomplates (MatTek) and exposed to 20% control or NMO patients' serum.Time-lapse photomicrographs of the living cells were captured at 15minute intervals for 6 hours using a Zeiss LSM510 microscope stage heldat 37° C. in 95% air/5% CO₂. Z-series imaging was performed for eachtime point.

Example 8—NMO-IgG Initiates Endolysosomal Degradation of AQP4

A prerequisite for IgG to affect organ-specific pathogenicity is itscapacity to bind to specific epitopes accessible on the surface ofliving target cells. When transfected target cells expressing surfaceAQP4 were exposed to a pool of NMO patients' sera (at 4° C. to restrictmembrane fluidity), IgG bound to the plasma membrane in a linear patternthat co-localized with GFP-AQP4. Serum IgG from control patients did notbind detectably to the cell surface and a control rabbit IgG specificfor AQP4 cytoplasmic epitopes did not bind unless the plasma membranewas permeabilized. NMO patients' serum IgG did not bind tovector-transfected control HEK-293 cells. Thus, NMO patients' IgG bindsspecifically to the extracellular domain of AQP4.

To evaluate the influence of this IgG on plasma membrane AQP4, thedistribution of GFP-AQP4 was recorded after exposure to patients' serumat 37° C. Control human serum had no effect on GFP-AQP4, but NMOpatients' sera containing AQP4-specific-IgG resulted in rapiddisappearance of GFP-AQP4 from the plasma membrane. The phenomenon'stemperature dependence is consistent with modulation of surface antigensthrough intermolecular cross-linking by bivalent IgG Serial confocalimages of living cells held at 37° C. for 16 hours in serum lackingAQP4-IgG were indistinguishable from images of untreated cells withrespect to GFP-AQP4 fluorescence intensity and localization. Cellsexposed to NMO patients' serum underwent a striking redistributionwithin 5 minutes. The homotetrameric structure of AQP4, with multiplerepeated epitopes in the extracellular domain, and the proximity ofindividual AQP4 homotetramers provide an optimal target for rapidsurface clearance from the plasma membrane by antigen-specific IgGPlasma membrane fading was accompanied by green fluorescent vesicleaccumulation in sub-plasmalemmal cytoplasm. After 20 minutes, GFP-AQP4aggregated in ring-like structures. At 5 hours, when plasma membranefluorescence was no longer detectable, these had coalesced into largecytoplasmic aggregates. Removal of the patients' serum at 16 hoursresulted in rapid reappearance of GFP-AQP4 in sub-plasmalemmal vesicles,presumably en route from the endoplasmic reticulum. By 4 hours, GFP-AQP4was visible in the plasma membrane and, by 12 hours, it had attainednormal surface density. When cycloheximide was added at the time ofwithdrawing patients' serum, no GFP-AQP4 was seen on the plasma membraneduring 12 hours of observation. This implies that the “reversal” of theautoantibody's effect was dependent on new protein synthesis.

The fate of GFP-AQP4 cleared from the plasma membrane after exposure toNMO patients' serum was followed by probing with an antibody specificfor an early endosome antigen. In the absence of serum, early endosomalvesicles did not colocalize with GFP-AQP4. However, after 30 minutesexposure to NMO patients' serum, early endosomal vesicles acquired greenfluorescence. Early endosomal vesicles did not acquire greenfluorescence when cells were exposed for 2 hours to control sera. Theseresults indicate that endocytosis initiated by the cross-linking of AQP4by IgG binding at the cell surface targets AQP4 to the endolysosomalpathway for degradation.

Example 9—NMO-IgG Initiates Complement Activation

Activation of the classical complement cascade is another potentiallypathogenic outcome of autoantibody binding to AQP4. This mechanism wouldincrease the permeability of microvascular endothelium, promoteinflammatory cell infiltration, and possibly inflict focal damage toastrocytic endfeet in central nervous system regions enriched in AQP4.Surface membranes of transfected cells were evaluated by indirectimmunofluorescence for deposition of the “C9neo” epitope created by thepolymerizaton of multiple perforin-like C9 molecules in assembling theterminal membrane attack complex of complement. In the presence of NMOpatients' sera and active human complement, C9neo was clearly visualizedin the plasma membrane of HEK-293 cells expressing AQP4, but was notvisualized in the absence of disease-specific serum, in the presence ofinactive human complement, or in control cells transfected withGFP-vector. These data indicate that NMO patients' sera contain anAQP4-specific antibody capable of activating the complement cascade.Reagents specific for human IgG subclasses revealed that AQP4-specificserum IgG was, in all NMO-IgG positive patients tested, exclusivelyIgG1, which is a major complement-activating IgG subclass in humans.

Preliminary experiments indicated that membrane lysis conditions had tobe rigorously controlled to evaluate functional evidence forantigen-specific complement activation by NMO-IgG on membranes of livingcells expressing AQP4 in high density. The GFP-AQP4 transfected targetcell line was a mixed population of GFP-positive (AQP4+) andGFP-negative (AQP4−) cells. In the presence of control human serum,phase microscopy revealed healthy AQP4+ and AQP4− cells in an adherentmonolayer. When the target cells were pre-exposed to disease-specificpatients' serum (on ice for 2 hours) before adding complement for 90minutes at 37° C. (conditions used for the C9neo visualization), allgreen cells disintegrated rapidly. Therefore, a standardized commercialpreparation of rabbit complement, in limited concentration, was used.The cells were pre-exposed to patient or control serum at 4° C. for 1hour before adding fresh or heat-inactivated complement. In the presenceof NMO patients' serum, adherent cells that visibly expressed GFP wereselectively lost. Provided that complement was active and present morethan 30 minutes, most AQP4+ cells were rounded and floating. Controlhuman serum did not visibly affect either adherent population regardlessof complement activity. Thus, the cytolytic activity of NMO patients'serum depended on both complement activity and AQP4 expression in targetcells.

Quantitation of target cell membrane lesioning by complement using flowcytometric analysis (indicated by permeability to the red dye, propidiumiodide) necessitated further attenuation of complement activation.Therefore, cells were pre-exposed to patients' (or control serum) atroom temperature for 30 minutes (rather than 1 hour at 4° C.) to allowsome reduction of AQP4 antigen density by antigenic modulation beforeadding the complement. Cells were then incubated with complement at 37°C. for only 30 minutes before harvesting for analysis of green and redfluorescence intensities. Exposure to active or inactive complement hadno effect on either AQP4+ or AQP4− cells in the presence of controlpatients' serum (FIG. 1). NMO patients' serum had no effect on membraneintegrity when complement was inactivated regardless of whether or notAQP4 was expressed in the target cells. However, with active complement,there was an average 5.6 fold (560%) increase in the membranepermeability of AQP4+ cells compared with AQP4− cells (FIG. 1). Thus,the cytotoxicity of NMO patients' serum depended on surface expressionof AQP4 and the availability of active complement.

Example 10—AQP4-Specific Autoantibodies are not IgM Class

Vasculocentric deposits of IgG IgM and complement are a hallmark ofhistopathological lesions found in central nervous system tissue of NMOpatients. Because pentameric IgM has five Fc effector domains, itactivates the complement cascade more efficiently than IgG, which hasonly a single Fc domain. Therefore, sera and cerebrospinal fluid of NMOpatients were analyzed for AQP4-specific IgM. By indirectimmunofluorescence evaluation on sections of AQP4-rich mouse tissues,none of 31 individual NMO patients' sera nor 28 NMO patients'cerebrospinal fluid specimens had detectable AQP4-specific IgM. The flowcytometry assay was used to test for functional evidence of acomplement-activating AQP4-specific IgM in serum from patients who wereeither positive or negative for AQP4 antibody of IgG class in the courseof clinical immunofluorescence evaluation. Only the AQP4 IgG-positivesera (pools 1-4) were cytotoxic for AQP4+ cells. This cytotoxic activitywas lost when IgG was depleted. Recovery of AQP4-specific complementactivation restored by IgG from the original positive serum pools wasproportional to the % total recovery of IgG from the Protein G-agarosebeads.

To determine, immunohistochemically, the frequency of IgG and IgMproduction in inflammatory central nervous system lesions of NMOpatients, accumulations of differentiated plasma cells were investigatedusing fluorochrome conjugated anti-human IgG and IgM class-specificreagents at limiting dilutions to avoid interference from backgroundextracellular IgG and IgM. The medullary lesion of the illustratedpatient contains abundant CD138-positive plasma cells and is in a regionnormally rich in AQP4. Plasma cells in the medullary lesion stainedbrightly for cytoplasmic IgG, but cytoplasmic IgM was not detected inany plasma cell. The immunoglobulin class specificity of the detectionantibody reagents was confirmed on control sections of human lymph nodetissue. Clusters of plasma cells exhibited bright cytoplasmic IgG andscattered plasma cells exhibited bright cytoplasmic IgM; very few cellswere dual-labeled. In summary, these data suggest that the IgM depositedwith IgG and complement products in AQP4-rich regions of NMO patients'central nervous system tissues is not AQP4-specific.

Example 11—AQP4-Enriched Astrocytic Processes Surround Nodes of Ranvierin Spinal Cord and Optic Nerve

Involvement of myelin in the pathology of NMO remains to be explained inthe context of AQP4. Freeze-fracture studies in the early 1970s defined“assemblies of particles” as an ultrastructural characteristic ofastrocytic endfeet surrounding axons at nodes of Ranvier. Theseassemblies correspond to recently identified orthogonal arrays formed byAQP4 homotetramers in transfected cells. The proximity of AQP4-specificimmunoreactivity in astrocytic membranes to nodes of Ranvier wasanalyzed by examining sections of mouse spinal cord and optic nervetissues by confocal fluorescence microscopy. Immune rabbit IgG (affinitypurified on AQP4 peptide) was used rather than NMO patient's serumbecause the latter is a complex mixture of multiple autoantibodies, andis not amenable to affinity purification because it binds only to nativeAQP4 and not to denatured membrane-extracted AQP4 or synthetic AQP4polypeptides. AQP4 was localized with respect to nodal regions bysimultaneously immunostaining sodium channels, which are denselyconcentrated in the nodal axonal membrane (or Casprl, an abundantparanodal protein), and glial fibrillary acidic protein (GFAP), thecytoplasmic intermediate filament of astrocytic processes. In bothspinal cord and optic nerve, AQP4 immunoreactivity was intense nearastrocytic elements and encircled periodic axonal segments expressingsodium channel immunoreactivity. The concentric paranodal AQP4immunoreactivity was brighter than the background mesh pattern ofastrocytic AQP4 characteristic of central nervous system white matterand was devoid of GFAP, which astrocytic foot processes lack. Theextracellular space surrounding astrocytic processes is enlarged in theregion of the axon segment bearing sodium channels. The juxtaposition ofintense AQP4 and Casprl immunoreactivities is consistent with extensionof the astrocytic foot process beyond the node of Ranvier into theparanode.

Part B Example 1—Cell Lines and Transgenic Constructs

Primary astrocytes isolated from cerebral cortices of P1-3 rats, and therat brain-derived O-2A progenitor cell line, CG-4, were grown aspreviously described (Louis et al., 1992, J. Neurosci. Res., 31:193-204;Garlin et al., 1995, J. Neurochem., 64:2572-80). The GFP-AQP4 constructand stably transfected human embryonic kidney cell lines (HEK-293) weredescribed previously (Lennon et al., 2005, J. Exp. Med., 202:473-7;Hinson et al., 2007, Neurology, 69:2221-31). AQP5 was amplified from ahuman salivary gland cDNA library, inserted into pEGFP-N1 vector(Clontech) and transfected (Fugene 6) HEK-293 cells with the parentvector or vector containing AQP5 transgenes. Stable clones weremaintained in DMEM supplemented with 10% bovine calf serum andantibiotics.

Example 2—Antibodies and Human Sera

Fluorochrome-conjugated goat IgGs were purchased from Molecular Probes(Alex-Fluor 546; human, mouse or rabbit IgG-specific, and Oregon Green;mouse IgG-specific) or Invitrogen (Cy5; rabbit IgG-specific), and goatIgGs monospecific for human IgG (TRITC-conjugated) or human IgM(FITC-conjugated) from Southern Biotechnology. Rabbit IgG specific for:AQP4 (residues 249-323) was purchased from Sigma, and rabbit IgGspecific for EAAT1 and EAAT2 was purchased from Santa CruzBiotechnologies (for Western blotting and immunoprecipitation) or AbCam(for immunofluorescence). Mouse monoclonal IgGs were purchased fromAbCam (anti-EEA1), Sigma (anti-GFAP conjugated to Cy3), TransductionLabs (anti-EAAT2) and Santa Cruz Biotechnologies (anti-GFP).De-identified sera from NMO and control patients were obtained, withMayo Clinic IRB approval, from the Neuroimmunology Laboratory,Department of Laboratory Medicine and Pathology, Mayo Clinic Rochester,Minn.

Example 3—Immunostaining

Cell lines grown on glass coverslips were rinsed in PBS and fixed in 4%PFA for 20 minutes at room temperature. After holding 30 minutes in 9%normal goat serum/0.1% triton X-100, the cells were held at 4° C.overnight in defined antibodies diluted in 10% normal goat serum, thenwashed in PBS, and held 60 minutes at room temperature in appropriatesecondary antibody diluted in 10% normal goat serum. After washing inPBS and mounting on a slide with ProLong® Gold DAPI antifade medium(Molecular Probes), fluorochrome-labeled cells were imaged using a ZeissLSM510 confocal microscope.

Sections of archival CNS tissues derived from control and NMO patients(5 μm, formalin-fixed and paraffin-embedded) were stained withhaematoxylin and eosin (HE), Luxol-fast blue-periodic acid-Schiff(LFB/PAS) or Bielschowsky silver impregnation. Avidin-biotin-basedimmunohistochemistry was performed without modification by incubatingtissue sections 1 hour with 10% normal goat serum, then holdingovernight at 4° C. with and without primary antibodies.

Example 4—Antigen Modulation Assay

Cells were dispensed onto glass coverslips coated with laminin (CG-4) orpoly-L-lysine (HEK-293 and primary rat astrocytes; Biocoat, BDBiosciences). After at least 48 hours, control or NMO serum was added to20% final concentration (with complement inactivated by holding 30minutes at 56° C.) and the cells were then processed forimmunofluorescence analysis.

Example 5—Complement Membrane Lesioning

Growth medium of confluent primary astrocytes growing in 6-well plateswas replaced with fresh medium containing 20% NMO or control sera. After15 minutes at room temperature, fresh or heat-inactivated complement(Low-Tox-H rabbit complement; Cedarlane Labs; final concentration 20%)was added and held for 40 minutes at 37° C., then processed for flowcytometric analysis.

Example 6—Glutamate Uptake Assay

Na+-dependent radiolabeled glutamate uptake was measured using primaryastrocytes grown to confluence in 24-well dishes (Lin et al., 2001,Nature, 410:84-8). After rinsing with 50 mM Tris-HCl and 320 mM sucrose,pH 7.4, control wells were incubated for 15 minutes with 10 mM unlabeledL-glutamate, then L-[3H]glutamate (0.1 μCi, GE Healthcare, specificactivity=55.0 μCi/nmol) was added in 25 mM NaHCO₃, 5 mM KCl, 1 mMKH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂) and 555 mM D-glucose, pH 7.4, with orwithout Na+(Kreb's buffer containing 120 mM NaCl, or 120 mM cholinechloride, in 25 mM Tris, 5 mM KCl, 1 mM KH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂)and 555 mM D-glucose, pH 7.4). Both buffers contained 40 μM unlabeledglutamate. After 5 mins at 37° C., the cells were transferred to 4° C.,washed extensively with PBS, lysed in 0.1 M NaOH and uptake ofradiolabeled glutamate was measured using a Beckman LS 6000SCscintillation counter. For all experimental conditions, quadruplicateassays were performed and glutamate uptake was calculated ascounts/min/well, with and without Na+.

To assay glutamate uptake in GFP vector and GFP-AQP4 stable HEK-293cells, an alternative HEK-293-optimized protocol was used (Jensen etal., 2004, Biochem. Pharmacol., 67:2155-27). EAAT2-specific transportwas defined as the difference in glutamate uptake between GFP vectorcells and GFP-AQP4 transfected cells.

Example 7—Isolation of RNA and RT-PCR Analysis

Total RNA was isolated from trizol-lysed cells, and double stranded cDNAwere generated using Superscript III First Strand Synthesis kit withrandom hexamer primers (according to Invitrogen protocols). To avoidamplifying contaminating genomic DNA, primer pairs were used that annealin adjacent exons: AQP4 (F5′-TGC ACC AGG AAG ATC AGC ATC G-3′ (SEQ IDNO:1) and R5′-CAG GTC ATC CGT CTC TAC CTG-3′ (SEQ ID NO:2)) and EAAT2(F5′-GGT GGA AGT GCG AAT GCA CGA CAG TCA TC-3′ (SEQ ID NO:3) and R5′-CCTCGT CTG GCG GTG GTG CAA CCA GGA C-3′ (SEQ ID NO:4)). Beta-actin wasamplified as a control.

Example 8—Immunoprecipitation and Western Blot

Protocols were as previously described (Lennon et al., 2005, J. Exp.Med., 202:473-477). Commercial antibodies (AQP4, EAAT1, EAAT2 or GFP, 2μg/mL), or human sera (a high titer pool of NMO patient or controlpatient sera, 30 μL/mL) were used as probes. Immune complexes releasedfrom protein G-agarose were resolved by electrophoresis (4 15% gradientpolyacrylamide, room temperature). Transblotted, blocked proteins wereexposed for 1 hour to IgG specific for: GFP (1:1,000), AQP4 (1:500),EAAT1 (1:200), actin (1:2000) or EAAT2 (1:200), then probed withhorseradish peroxidase-conjugated goat anti-mouse IgG or goatanti-rabbit IgG and detected bound IgG autoradiographically (SuperSignalWest Pico Chemiluminescence, Pierce).

Example 9—Statistical Analysis

Significance was calculated using Student's t-test (2-tailed).

Example 10—NMO-IgG Binding to Primary Astrocytes Induces AQP4 Modulationand Complement Activation

AQP4 distribution was monitored after applying NMO or control serum tocerebral astrocytes. Serum containing NMO-IgG but not control serum,induced rapid down-regulation of surface AQP4. AQP4 coalesced incytoplasmic vesicles, reminiscent of those observed inGFP-AQP4-transfected non-neural cells exposed to NMO-IgG.

The complement-activating capacity of NMO-IgG binding to astrocyticAQP4, determined by including complement with NMO or control serum, wasevaluated and the cells processed for flow cytometric analysis ofpropidium iodide influx. In the presence of NMO-IgG and activecomplement, plasma membrane permeability increased approximately 2 fold(p<0.007; FIG. 2A). With heat-inactivated complement, no discernibleeffect was observed with control serum or NMO serum (FIG. 2A).Activation of early complement components by IgG binding to theextracellular domain of AQP4 in astrocytes would increase CNSmicrovascular endothelial permeability and promote inflammatory cellinfiltration. Assembly of the final membrane attack complex mightfocally damage endfeet, where AQP4 is expressed most highly. However,rich endowment of astrocytic membranes with complement regulatoryproteins, as has been reported, may explain their relative resistance toAQP4-IgG-dependent lysis in this study, in comparison toAQP4-transfected HEK-293 cells.

Example 11—NMO-IgG Attenuates Na+-Dependent Glutamate Uptake inAstrocytes

Na+-dependent glutamate transport in astrocytes exposed to NMO orcontrol sera (FIG. 2B) was compared. Uptake of L-[³H]glutamate wasminimal (<200 counts/minute) in the absence of Na+ or when excessunlabeled glutamate was present. In the presence of Na+, L-[³H]glutamateuptake was increased to approximately 3000 counts/minute in cellsunexposed to serum or exposed to control patient serum (FIG. 2B). Theseresults are consistent with the uptake of L-[³H]glutamate beingNa+-dependent and glutamate-specific. When serum containing NMO-IgG wasadded in these experimental conditions, L-[³H]glutamate uptake wasreduced by approximately 50% (p<0.0003; FIG. 2B).

Example 12—NMO-IgG Down-Regulates Both EAAT2 and AQP4 from the Surfaceof Differentiated Type-2 Astrocytic Cells

Attenuation of astrocytic L-[³H]glutamate uptake after exposure toNMO-IgG parallels loss of AQP4 protein. To investigate the possibilitythat EAAT2 protein may be lost secondary to loss of surface AQP4,EAAT2-specific IgG was used to follow the fate of EAAT2 after exposingastrocytes to NMO-IgG Consistent with previous reports, it was confirmedthat EAAT1 and EAAT2 glutamate transporter levels in primary ratastrocyte membranes are too low to detect by immunofluorescencestaining. Therefore, EAAT2 expression was investigated in thebipotential glial cell line, CG-4, derived from O2-A progenitor cells inthe developing rat CNS. In prescribed culture conditions (Louis et al.,1992, supra), CG-4 cells differentiate into type-2 astrocytes. Inproliferation-promoting medium (containing bFGF and PDGF), the cells(CG4-PM) lack GFAP intermediate filaments. However, in medium withgrowth factors replaced by a high concentration of fetal bovine serum,type-2 astrocyte differentiation was evident in the cells (CG4-AM) bymorphology and GFAP-immunoreactivity. In cells grown 7 days in astrocytedifferentiation medium, plasma membrane expression of both AQP4 andEAAT2 was enhanced. However when NMO serum was added to CG4-AM cells atday 7, both AQP4 and EAAT2 were depleted from the plasma membrane.Addition of control sera in identical conditions did not discerniblyaffect expression of either AQP4 or EAAT2. Concomitant loss of bothEAAT2 and AQP4 plausibly explains the reduced glutamate transport thatwas observed in cultured astrocytes exposed to NMO-IgG.

Example 13—EAAT2 Expression is Up-Regulated when AQP4 Protein Expressionis Induced in Non-Neural Cells

AQP4 and EAAT2 are both enriched in the astrocytic endfoot membrane.This finding that both AQP4 and EAAT2 are depleted from plasma membranesof cultured astrocytes exposed to NMO-IgG is consistent with the notionthat EAAT2 expression depends on AQP4 expression. To further evaluatethe relationship between AQP4 and glutamate transport, the HEK-293non-neural cell line were studied, comparing EAAT2 expression in cellsstably expressing GFP, GFP-AQP4, or a non-neural AQP, AQP5-GFP. EAAT1was readily detected in the plasma membranes of cell lines transfectedwith either GFP vector or GFP-AQP4. EAAT2 was not detected in GFP vectortransfected cells, but EAAT2 was strikingly upregulated in the plasmamembrane of cells transgenically expressing either AQP4 or AQP5.

To determine whether this observed increase in membrane EAAT2 andfunctional glutamate transport might reflect an increase in EAAT2 genetranscription or protein expression, EAAT2 transcripts were examined byRT-PCR. The three transfected HEK-293 lines expressed EAAT2 transcriptsat similar levels. Western blot analyses supported theimmunofluorescence observations that transgenic EAAT2 protein expressionis upregulated when AQP4 or AQP5 is expressed in HEK-293 cells bycomparison with cells transfected with vector alone. Expression of EAAT2protein on the surface of cells expressing AQP might be increasedthrough upregulated mRNA translation or, alternatively, through apost-translational modification increasing EAAT2 protein stability ortrafficking to the plasma membrane. These complementary observationsaccord with reports that EAAT2 protein expression is restricted toastrocytes, despite ubiquitous expression of EAAT2 mRNA. It wasconcluded that restriction of EAAT2 expression to the plasma membrane ofastrocytes is determined by dependence on astrocytic AQP expression.

These results confirmed that upregulated EAAT2 protein in GFP-AQP4transfected cells was functional by demonstrating that GFP-AQP4 cellsimported 2-3 fold more glutamate via the Na+-dependent pathway relativeto GFP vector cells (p<0.0002; FIG. 3). It is noteworthy that glutamatetransport in Na+-containing buffer was unchanged in GFP vector cellscompared to GFP-AQP4 (FIG. 3). It was anticipated that constitutivelyexpressed EAAT1 in both cell lines would confer higher glutamatetransport in GFP vector cells in Na+-containing buffer than in bufferlacking Na+. However, uptake of glutamate was unaffected in the presenceof Na+, suggesting that EAAT1 expressed constitutively in these cells isnot functional.

Example 14—Plasma Membrane Loss of EAAT2 Following Exposure to NMO Serumis EAAT2-Selective and is Dependent on the Presence of Both AQP4 Proteinand NMO-IgG

The data reported herein indicate that the concentration of AQP4 proteinin the plasma membrane and Na+-dependent glutamate transport are bothreduced by exposure of primary astrocytes to NMO-IgG. These observationsin differentiated CG-4 type-2 astrocytes suggest that the effect onglutamate transport in primary astrocytes is due to plasma membrane lossof EAAT2. To further investigate the association between AQP4 and EAAT2,the effect of NMO and control sera on both EAAT1 and EAAT2 wasevaluated. Control serum did not appreciably affect localization orexpression of the EAAT2 transporter. However, serum containing NMO-IgGinduced rapid surface down-regulation of both GFP-AQP4 and EAAT2. Highermagnification revealed apparent co-localization of EAAT2 and AQP4 inearly endosomal vesicles, to which AQP4 translocation after exposure toNMO serum was previously demonstrated. The localization of AQP4 and theearly endosome antigen-1 marker was evaluated after exposing the cellsto NMO serum for 10 minutes. The white color that resulted when theimages were merged suggested partial co-localization of AQP4 and EAAT2in early endosomes. However, separate vesicles in close proximity oroverlapping in the z axis would yield the same result.

The possibility that NMO serum might contain EAAT2-specific IgG inaddition to AQP4 IgG was excluded by testing the effect of NMO serum onplasma membrane expression of EAAT2 in HEK-293 cells transfected withAQP5-GFP. Those cells express both EAAT1 and EAAT2, but are devoid ofAQP4. Exposure to NMO-IgG did not affect the distribution of EAAT1 orEAAT2 in AQP5-GFP cells.

To investigate the specificity of EAAT2 down-regulation, the effect ofNMO-IgG on the EAAT1 glutamate transporter, which is expressedconstitutively in non-neural HEK-293 cells, was evaluated. NMO patientsera did not appreciably affect EAAT1 expression in these cells, incontrast to the loss of EAAT2 from the plasma membrane. These resultsimply a specific association between AQP4 and EAAT2 that does not existfor EAAT1. The rapid down-regulation of EAAT2 and AQP4 induced byNMO-IgG in GFP-AQP4-expressing cells and co-localization of bothproteins in cytoplasmic endocytotic vesicles within 10 minutes isconsistent with a direct effect of IgG on a surface macromolecularcomplex. The reduction in astrocytic glutamate transport accompanyingAQP4 down-regulation after exposure to NMO-IgG further supports the ideathat AQP4 and EAAT2 are associated functionally in the plasma membrane.

Example 15—AQP4 and EAAT2 Co-Immunoprecipitate

EAAT2 and EAAT1 are enriched in separate microdomains of the astrocyticplasma membrane; EAAT2 is enriched in regions that highly express AQP4.The data reported herein suggest that EAAT2 and AQP4 exist as amacromolecular complex. When exposed to NMO-IgG both are translocatedfrom the plasma membrane to an endolysosomal-targeted population ofcytoplasmic vesicles. To evaluate potential physical interaction,GFP-AQP4 transfected cells was solubilized and the lysates were probedwith EAAT2-IgG. Addition of protein G-agarose captured both EAAT2 andAQP4. Similar data was obtained using independent IgGs recognizingdifferent EAAT2 epitopes. As a specificity control, results obtainedusing EAAT1-IgG as a probe for the cell lysates was compared. Incontrast to EAAT2-IgG EAAT1-IgG did not pull down AQP4. These resultssupport the existence of AQP4 and EAAT2 as a macromolecular complexindependent of EAAT1.

Example 16—Co-Localization of AQP4 and EAAT2 in CNS Tissue

The observations in primary astrocytes, type-2 differentiated CG-4 cellsand transfected non-neural HEK-293 cells indicate that the interactionof NMO-IgG with AQP4 induces at least three possible outcomes, eachpotentially pathogenic: complement activation, down-regulation of AQP4,and coupled down-regulation of the EAAT2 glutamate transporter. Theimmunohistochemical analysis of non-pathologic human CNS tissue (bothcortical and spinal cord) revealed that EAAT2, but not EAAT1, normallyco-localizes with AQP4 in gray matter astrocytes and that EAAT2 is mostenriched in spinal cord gray matter. These findings were reproducible.

Example 17—NMO Spinal Cord Lesions Lack Both AQP4 and EAAT2

Loss of AQP4 is a distinctive finding in both early and late lesions ofNMO. NMO spinal cord tissue of normal appearance expresses normal levelsof APQ4 and EAAT2 and lacks evidence of complement deposition. LesionedNMO spinal cord gray matter contrasts to normal appearing gray matter byexhibiting markedly reduced EAAT2, in addition to AQP4 loss anddeposition of complement activation products. Together, these findingsare consistent with the absence of EAAT2 staining being a biologicalphenomenon within the NMO lesion. EAAT2 loss may partially account forthe destructive involvement of spinal cord gray matter which ischaracteristic of NMO. Lesions in MS CNS tissues are typically notnecrotic. The marked loss of EAAT2 described herein parallels loss ofAQP4 in lesioned NMO spinal cord tissue and contrasts with the increasesin EAAT2 and AQP4 reported in both active and chronic MS lesions. It wasconcluded that differences in regulation of glutamate homeostasisfurther distinguish NMO from classical MS.

Example 18—Concluding Remarks

The data presented herein from studies of living astrocytes, patient andcontrol sera, normal human spinal cord tissue and spinal cord tissuesfrom a patient with typical NMO, both non-lesioned and lesioned, providethe first evidence supporting a pathogenic role for NMO-IgG indisrupting glutamate transport. Because astrocytes are relativelytolerant to increased glutamate concentrations, disruption of glutamatehomeostasis by NMO-IgG has particular excitotoxic potential for neuronsand oligodendrocytes. A focal increase of extracellular glutamate levelssecondary to NMO-IgG-induced down-regulation of AQP4 may suffice toinjure or kill oligodendrocytes that express calcium-permeable glutamatereceptors. Oligodendrocytes in the spinal cord and optic nerve, whichare principal sites of demyelination in NMO, are particularly sensitiveto changes in glutamate concentration. Modest elevation of extracellularglutamate concentration renders oligodendrocytes additionallysusceptible to Ig-independent complement attack. The potentialpathogenic sequelae demonstrated in this study for NMO-IgG binding toAQP4-rich membranes in primary astrocytes are both competing andcooperative. Depletion of AQP4 water channels in the plasma membranewould disrupt water homeostasis and promote edema. If complement werelacking, the consequences of impaired glutamate transport would beparticularly deleterious for oligodendrocytes and neurons.

Outcomes of therapies directed at glutamate receptors have beenunimpressive for neurodegenerative conditions where glutamate toxicityhas been implicated in disease progression. However, demonstrating thatthe major astrocytic glutamate transporter, EAAT2, exists in amacromolecular complex with the AQP4 water channel and is down-regulatedby AQP4-specific autoantibodies that are restricted to patients with theNMO-spectrum of CNS inflammatory autoimmune demyelinating disorders hasunanticipated pathogenic implications for glutamate toxicity as acentral mechanism in a spectrum of disorders which are commonly mistakenfor MS. NMO is now recognized as a potentially reversible IgG-mediatedattack on astrocytic water channels. The results that were obtained fromstudies of serum and spinal cord tissue of patients with NMO holdpromise for novel therapeutic strategies for the management ofNMO-spectrum disorders. For example, it might be feasible to amelioratetissue damage in both grey and white matter if therapeutic upregulationof EAAT2 can be achieved in patients whose neurological dysfunction isattributable to AQP4 autoimmunity.

Example 19—Prognostic Utility of Functional Assays for NMO/AQP4-IgG

Preliminary data (Table 1) revealed a lack of correlation between serumlevels of NMO-IgG (by immunofluorescence assay) and attack severity andbetween NMO-IgG immunofluorescence titers and complement activationquantified by flow cytometric analysis comparison with cells exposed tohealthy control subjects' serum. In contrast, functional assaysquantitating the outcome of NMO-IgG binding to cells expressing AQP4have revealed significant correlation between the severity of an acuteNMO attack and activation of complement. For the purposes of thesestudies, attacks were classified as “mild” when visual or limbimpairment was minimal or when complete recovery occurred (Patients1-6), and as “severe” when the attack resulted in residual blindness(from optic neuritis) or paralysis (from spinal cord inflammation)(Patients 7-12).

TABLE 1 NMO Complement-dependent Individuals whose sera Attack lesioningof AQP4 + were tested Severity IF Titer HEK293 cells (%) Patients withacute attack  1 mild 1920 10  2 mild 240 15.9  3 mild 960 7.8  4 mild960 19.1  5 mild 7680 12.3  6 mild 1920 19.2  7 severe 7680 59.8  8severe 960 20.5  9 severe 1920 27.7 10 severe 3840 51.8 11 severe 1536060.1 12 severe 7680 55.7 Healthy Controls  1 — Negative 7.5%  2 —Negative 8.2%  3 — Negative 9.1%  4 — Negative 8.0%  5 — Negative 11.6% 6 — Negative 7.7%  7 — Negative 7.6%  8 — Negative 9.9%  9 — Negative7.6% Multiple Sclerosis Controls MS 1 — Negative 9.2% MS 2 — Negative11.9% MS 3 — Negative 10.5% MS 4 — Negative 10.7% MS 5 — Negative 12.5%MS 6 — Negative 14.3% MS 7 — Negative 13.2% MS 8 — Negative 14.8% MS 9 —Negative 14.3%

Measures of complement activation by NMO-IgG were significantly(p<0.005) associated with attack severity classification (mild orsevere) but NMO-IgG titers were not (p>0.05). Quantitative functionaleffects of patient serum on AQP4-expressing cells can be assayed oncultured astrocytes, astrocytic cell lines or transfected non-neuralcells. Correlation with clinical variables provides informationpertinent to anticipated outcome of attack (e.g., relapse risk, attackseverity (e.g., vision loss, paralysis), or extent of recovery).

These data support an algorithmic approach for the serologicalevaluation of patients with inflammatory autoimmune demyelinatingdisorders of the central nervous system (FIG. 4). Seropositivity ininitial immunochemical screening assays for NMO-IgG supports thediagnosis of an NMO spectrum disorder. Further testing of seropositivepatients using functional assays (as demonstrated by the complementactivation assay described above; and also by AQP4 and EAAT2 transportermodulating assays described herein) is anticipated to provide prognosticinformation guiding therapeutic decision making. It is also observedthat AQP4-modulating assays are sufficiently sensitive to detect NMO-IgGin some clinically proven cases that are negative by immunochemicalassays.

It is possible that diverse serological effects may occur in individualpatients. For example, one patient's disease severity may correlate wellwith measures of AQP4 complement activation while another may correlatewith measures of AQP4/EEAT2 downregulation. In future clinical practice,the choice of treatment for individual patients with NMO spectrumdisorders may be determined by the individual's serological profile infunctional assays of AQP4/NMO-IgG. For example, patients whose sera arehighly complement activating may be ideal candidates for complementinhibitory therapies, or patients whose EAAT2 levels are significantlyreduced may be ideal candidates for glutamate receptor antagonisttherapies.

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

1. (canceled)
 2. A method of treating an individual that has a disorderrelated to aquaporin-4 (AQP4) autoimmunity, said method comprisingadministering an anti-05 antibody to an individual identified as beingserologically positive for AQP4-IgG autoantibody.
 3. The method of claim2, wherein said disorder is neuromyelitis optica (NMO) or anNMO-associated disease.
 4. The method of claim 2, wherein said anti-05antibody is administered orally.
 5. The method of claim 2, furthercomprising administering a glutamate receptor antagonist.
 6. The methodof claim 5, wherein said glutamate receptor antagonist is selected fromthe group consisting of 1-amino-3,5-dimethyl-adamantane,1-aminoadamantane, (+)-3-methoxy-17-methyl-(9α,13α,14α)-morphinan,17-methyl-9a,13a,14a-morphinan-3-ol,2-(2-chlorophenyl)-2-methylamino-cyclohexan-1-one,1-(1-phenylcyclohexyl)piperidine,(±)cis-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl) cyclohexanolhydrochloride, and 6-(Dimethylamino)-4,4-diphenylheptan-3-one.
 7. Themethod of claim 5, wherein said glutamate receptor antagonist is1-amino-3,5-dimethyl-adamantane, 1-aminoadamantane.
 8. The method ofclaim 2, further comprising monitoring the individual for a decrease inthe severity of said disorder.
 9. A method of treating an individualthat has a disorder related to AQP4 autoimmunity, said methodcomprising: (a) identifying an individual as being serologicallypositive for AQP4-IgG autoantibody; and (b) administering an anti-05antibody to the individual.
 10. The method of claim 9, wherein saiddisorder is NMO or an NMO-associated disease.
 11. The method of claim 9,wherein said anti-05 antibody is administered orally.
 12. The method ofclaim 9, further comprising administering a glutamate receptorantagonist.
 13. The method of claim 12, wherein said glutamate receptorantagonist is selected from the group consisting of1-amino-3,5-dimethyl-adamantane, 1-aminoadamantane,(+)-3-methoxy-17-methyl-(9α,13α,14α)-morphinan,17-methyl-9a,13a,14a-morphinan-3-ol,2-(2-chlorophenyl)-2-methylamino-cyclohexan-1-one,1-(1-phenylcyclohexyl)piperidine,(±)cis-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl) cyclohexanolhydrochloride, and 6-(Dimethylamino)-4,4-diphenylheptan-3-one.
 14. Themethod of claim 13, wherein said glutamate receptor antagonist is1-amino-3,5-dimethyl-adamantane, 1-aminoadamantane.
 15. The method ofclaim 9, further comprising monitoring the individual for a decrease inthe severity of said disorder.